Research in Progress Currently, there are Four main ongoing projects in the lab: The first project is focused on elucidating mechanistic details of the interaction between type II topoisomerases and DNA. In particular, we have a longstanding interest in the complex interplay between DNA topology and the binding and activity of type II topoisomerases. One aspect of this interaction concerns the ability of type II topoisomerases to relax the topology of DNA to below equilibrium values. In vivo these topoisomerases are responsible for unlinking replicated chromosomes prior to cell division. Since even a single link between sister chromosomes can prevent division and induce cell death, it is important that these enzymes preferentially unlink rather than link DNA molecules. In vitro it was shown that this is the case, but the mechanism remains a mystery. We continue to experimentally test mechanistic models for below equilibrium topology simplification. Another aspect of topology-dependent activity of type II topoisomerases is their ability to distinguish the chirality of the supercoiling. This review period in collaboration with Neil Osheroff from Vanderbilt university we demonstrated a dramatic 50-fold or more increase in the relaxation of positive supercoils over the introduction of negative supercoils by the Bacillus anthracis DNA gyrase. This finding has implications for the targeting of antibiotics against bacterial gyrases, and possibly impacts the regulation of negative supercoiling of the genome in bacteria. More generally, the Bacillus anthracis DNA gyrase is the fastest type II topoisomerase discovered to date and its impressive rate alters our understanding of what limits the enzymatic rate of these enzymes. We have recently initiated a collaborative project with Anthony Maxell of the John Innes Center in the UK investigating the activity and topological selection by topoisomerase VI, which is a type IIb topoisomerase. The type IIb enzymes are structurally related to the type IIa enzymes, but they lack a key element (the C-terminal gate) that is believed to contribute to the directionality of the type IIa enzymes. We are using a combination of single-molecule and ensemble methods to probe the basic strand passage mechanism of this topoisomerase VI from Methanosarcina Mazei. Our findings from this topoisomerase IIb may have important ramifications for the topoisomerase VI enzymes from the malaria parasite Plasmodium falciparum, and from plants. Small molecule inhibitors or poisons for topoisomerase VI are actively sought for potential malarial treatment and herbicides. The second project is focused the mechanisms underlying multi-enzyme complex activity. RecQ helicases and topoisomerase III have been shown to functionally and physically interact in organisms ranging from bacteria to humans. Disruption of this interaction leads to severe chromosome instability; however the specific activity of the enzyme complex is unclear. Analysis of the complex is complicated by the fact that both the helicase and the topoisomerase individually modify DNA. In collaboration with Mihaly Kovacs at Etovos University, Hungry, we are using single-molecule measurements of DNA unwinding and unlinking to elucidate the detailed of RecQ helicase activity alone and in the presence of Topo III. These experiments will pave the way for experiments in which the activity and the association state of single enzymes and complexes will be assayed simultaneously using a combination of single molecule manipulation and single molecule visualization techniques. Working towards the overarching goal of understanding the mechanistic basis for the chromosome maintenance activities of the RecQ-Topo III complex, we have recently dissected the functional roles of specific and conserved protein domains in both the bacterial RecQ and in the human ortholog, Blooms syndrome helicase. We identifieda novel DNA geometry-dependent binding mode of RecQ helicases mediated by a specific domain. We further establish the importance of this domain for proper resolution of recombination intermediates both in vitro and in vivo. In follow up work, we have determined the mechanism through which RecQ unwinds DNA and how this mechanism leads to the coordinated binding of key accessory domains involved in preserving genomic stability. We recently demonstrated that RecQ helicase can remove single stranded binding protein (SSB) from single-stranded DNA and we elucidated the molecular mechanism through which this process is mediated. This work contributes to our understanding of the putative role of SSB or the eukaryotic homolog replication protein A (RPA) plays in the activity within the complex of a RecQ helicase, Topoisomerase III, and SSB or RPA. The third related project, in collaboration with Yves Pommier in NCI, is focused on the mechanisms of supercoil relaxation by human type IB topoisomerases, and in the effects of chemotherapy agents that inhibit type IB topoisomerases. Type IB topoisomerases are essential enzymes that relax over wound (positively supercoiled) DNA generated ahead of the replication machinery during DNA synthesis. Type IB topoisomerases are also important chemotherapy targets. Potent chemotherapy agents that specifically inhibit type IB topoisomerases are currently in clinical use and additional agents are in development. We are using single-molecule magnetic-tweezers based assays to measure the activity of individual type IB topoisomerases and the effects of chemotherapy agents on the activity. These experiments provide molecular level details of the supercoil relaxation process and how it is influenced by the degree of DNA supercoiling, the tension on the DNA, and the presence of specific chemotherapy agents. In recent work, we have also characterized two human mitochondrial topoisomerase variants associated with single nucleotide polymorphisms that are correlated with certain cancer types in the NCI cancer data base, and a point-mutant of mitochondrial topoisomerase associated with mitochondrial disease. The single-molecule measurements revealed specific alterations in the activity of these topoisomerase mutants at relatively high supercoiling levels that were not detectable in conventional biochemical assays of topoisomerase activity. The fourth project involves the role of DNA topology on the identification and repair of DNA damage. We recently established that a single mismatched base in 6 kb of DNA will preferentially localize the tip of a plectoneme at the mismatch. This experimental finding was theoretically extended in collaboration with John Marko at Northwestern University. These experimental and theoretical results indicate that supercoiling of DNA can contribute to the localization and identification of mismatches or other DNA damage by repair enzymes that recognize sharply bent DNA with a flipped-out base, both of which are favored when the damaged site is localized at the tip of a plectoneme in supercoiled DNA. We are further extending these results via multiscale simulations of DNA containing mismatches in collaboration with Siddhartha Das in the Mechanical Engineering department at the University of Maryland. These projects have been enabled by the development of a unique magnetic tweezers instrument that affords high spatial and temporal resolution measurements of DNA topology combined with real-time computer control and position stabilization. The ongoing development and improvement of this magnetic tweezers instrument represents a sustained research endeavor. We have recently added a total internal reflection microscope modality to the magnetic tweezers instrument that permits single-molecule fluorescence measurements in conjunction with single-molecule manipulation via the magnetic tweezers.